Regulation of protein expression by hypoxia is complex and is mediated through both translational and post-translational controls. Post- translational regulation by hypoxia is an underappreciated means of regulating protein expression and is best exemplified by the alpha subunit for the HIF-1 transcription factor. Hypoxia is very unique in that it has developed a means of modulating gene expression by regulating the post- translational stabilization of a protein. Genetic and molecular analysis of HIF-1 alpha indicates that its hypoxia responsiveness is governed by two different domains located in its carboxy terminal region. One domain regulates its stabilization under hypoxic conditions (ODD) and a second domain governs its hypoxia inducible transactivation activity. Importantly, addition of the ODD to heterologous proteins can result in their enhanced degradation under aerobic conditions and increased stabilization under hypoxic conditions. Thus, one goal of the grant is to devise a scheme to regulate protein stability (and as a consequent protein activity) by changes in the oxygen environment of a tumor. However, such a strategy would have low clinical translational potential if there is not an efficient means of transducing the protein into a high percentage of tumor cells in vivo. Fortunately, a new approach to protein transduction is available that has great promise for in vivo therapeutics This approach involves the transfer of proteins into cells through their tethering to the HIV TAT protein. To date over 50 proteins ranging from 10 to 110 kd have been transferred into cells using TAT. The combination of TAT transduction of proteins under the regulation of the HIF-1 alpha ODD is a powerful means of modulating protein expression in tumor tissue and sparing normal tissue based on the well validated premise that hypoxia is not found in normal tissue. In this proposal we will test four hypotheses that will determine whether such an approach can be developed for enhanced anti-tumor therapy: 1) Proteins can be made unstable under aerobic conditions by the addition of the HIF-1a oxygen degradation domain, 2) protein transduction using the HIV-TAT protein transduction domain (PDT) will be an efficient means of introducing proteins can be enhanced in tumor cells that are oxygen labile into cells in vitro, 3) tumors in vivo and 4) Stabilization of ODD transduced proteins can be enhanced in tumors cells that possess adys Ras/PI-3K signaling pathway or have lost the VHL tumor suppressor gene.